PROJECT SUMMARY Recent developments in mass spectrometry (MS)-based metabolomic approaches are helping to establish lipid molecules as critical mediators of signal transduction in the nervous system. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), for example, serves as a key metabolic hub connecting the endogenous cannabinoid system (ECS) and pro-inflammatory eicosanoid production in the brain. In this manner, 2-AG signaling pathways modulate pain, anxiety, neuroinflammation, and neurodegeneration and have thus attracted considerable pharmaceutical interest for the treatment of nervous system diseases. Global increases in brain 2-AG, however, can also cause a range of undesirable side-effects. The multiplicity and differential distribution of 2-AG metabolic enzymes may provide a means to target specific brain 2-AG stores to fully capitalize on the development of 2-AG-based therapeutics with acceptable safety profiles. We hypothesize that the inhibition of distinct 2-AG biosynthetic and degradative enzymes will modulate the ECS/eicosanoid signaling network in specific brain cellular compartments to produce anti-inflammatory and neuroprotective effects, while limiting detrimental consequences observed with global ECS agonism or antagonism. This proposal describes experiments that leverage newly generated tools to selectively disrupt 2-AG metabolic enzymes together with the power of MS-based lipidomics to define novel enzymatic targets for the development of eCB-based therapeutics. First, we will map the cellular anatomy of 2-AG degradation pathways in the mouse brain. Specifically, we will use targeted and untargeted lipidomic profiling to examine the compartmentalization of the major brain 2-AG hydrolases (MAGL, ABHD6 and ABHD12) in neurons vs. glia, as well as to characterize cell type-specific metabolic alterations following genetic deletion of these enzymes. Second we will determine whether the inhibition of diacylglycerol lipases (DAGL) a and b, the two main 2-AG biosynthetic enzymes in the brain, can selectively modulate the brain ECS/eicosanoid network. By taking advantage of DAGLa or b knockout mice, we will map the cellular distribution of these two enzymes in the nervous system and assess how their inactivation alters pools of eCBs/eicosanoids basally and in experimental paradigms of neuroinflammation. Finally, we will carry out MS-based metabolic imaging of the anatomical distribution of 2-AG in the brain. We will use a novel Nanostructure-initiator Mass Spectrometry (NIMS) imaging platform to characterize the distribution of 2-AG levels in different brain regions basally and after inhibition of 2-AG metabolic enzymes. In summary, this project will push the frontiers of applying cutting- edge lipidomic technologies to characterize the cellular and anatomical compartmentalization of the ECS/eicosanoid signaling network and its role in brain intercellular communication, as well as aid the development of novel eCB-based therapeutics for the treatment of nervous system diseases.